NKCC2 surface expression in mammalian cells - down-regulation by novel interaction with aldolase b
NKCC2 surface expression in mammalian cells - down-regulation by novel interaction with aldolase b
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DOI：
10.1074/jbc.m700195200
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发表时间：
2007-11-16
影响因子：
4.8
通讯作者：
Laghmani, Kamel
中科院分区：
文献类型：
--
作者：
Benziane, Boubacar;Demaretz, Sylvie;Laghmani, Kamel
顶端对布美他尼敏感的Na+-K+-2Cl(+)共转运蛋白NKCC2是肾脏肥厚升支的主要盐分转运途径。NKCC2的表面表达受细胞内蛋白运输的调节。然而，参与NKCC2细胞内运输的蛋白质伙伴仍不清楚。此外，由于在哺乳动物细胞中表达NKCC2蛋白的困难，旨在了解NKCC2翻译后调控的研究一直受到阻碍。在此，我们通过标记NKCC2的N-末端结构域，在肾上皮细胞中表达了NKCC2蛋白。为了深入了解NKCC2转运的调控，我们以NKCC2的C末端尾巴为诱饵，筛选了NKCC2与酵母双杂交系统的相互作用伙伴。醛缩酶B是一种新的优势相互作用蛋白。实时荧光定量聚合酶链式反应显示肾脏粗大上肢有醛缩酶B的表达。免疫共沉淀和免疫共定位实验证实了NKCC2-醛缩酶在肾细胞中的相互作用。生物素化分析表明，醛缩酶共表达降低了NKCC2的表面表达。在醛缩酶底物1，6-二磷酸果糖存在下，醛缩酶的结合被破坏，共表达的醛缩酶对NKCC2细胞表面水平没有进一步的影响。最后，功能研究表明，醛缩酶诱导的NKCC2在质膜上的下调与其转运活性的降低有关。综上所述，我们确定醛缩酶B是一种新的NKCC2结合伙伴，在NKCC2表面表达的调控中发挥关键作用，从而揭示了一种新的调控共转运体细胞内运输的机制。此外，NKCC2蛋白在哺乳动物细胞中的表达及其通过蛋白质-蛋白质相互作用的调节，可能为研究细胞生物学和共转运蛋白的转录后调控开辟新的重要途径。
Apical bumetanide-sensitive Na+-K+-2Cl(+) co-transporter, termed NKCC2, is the major salt transport pathway in kidney thick ascending limb. NKCC2 surface expression is subject to regulation by intracellular protein trafficking. However, the protein partners involved in the intracellular trafficking of NKCC2 remain unknown. Moreover, studies aimed at understanding the post-translational regulation of NKCC2 have been hampered by the difficulty to express NKCC2 protein in mammalian cells. Here we were able to express NKCC2 protein in renal epithelial cells by tagging its N-terminal domain. To gain insights into the regulation of NKCC2 trafficking, we screened for interaction partners of NKCC2 with the yeast two-hybrid system, using the C-terminal tail of NKCC2 as bait. Aldolase B was identified as a dominant and novel interacting protein. Real time PCR on renal microdissected tubules demonstrated the expression of aldolase B in the thick ascending limb. Co-immunoprecipitation and co-immunolocalization experiments confirmed NKCC2-aldolase interaction in renal cells. Biotinylation assays showed that aldolase co-expression reduces NKCC2 surface expression. In the presence of aldolase substrate, fructose 1,6-bisphosphate, aldolase binding was disrupted, and aldolase co-expression had no further effect on the cell surface level of NKCC2. Finally, functional studies demonstrated that aldolase-induced down-regulation of NKCC2 at the plasma membrane was associated with a decrease in its transport activity. In summary, we identified aldolase B as a novel NKCC2 binding partner that plays a key role in the modulation of NKCC2 surface expression, thereby revealing a new regulatory mechanism governing the co-transporter intracellular trafficking. Furthermore, NKCC2 protein expression in mammalian cells and its regulation by protein-protein interactions, described here, may open new and important avenues in studying the cell biology and post-transcriptional regulation of the co-transporter.